| Background:Diabetic kidney disease,one of the serious microvascular complications in diabetes,has caused a tremendous burden on families and society.Glomerular hypertrophy and ultrafiltration are early lesions of diabetic kidney disease,and mesangial cells are at the center of the pathogenesis of these changes.At the same time,our research group found in previous studies that endoplasmic reticulum stress plays an important role in the pathogenesis of diabetic kidney disease,and glucose regulatory protein 78(GRP78)is its key marker protein.It is worth noting that mitochondria and endoplasmic reticulum are very important organelles in cells,and there is a close relationship between structure and function between them.A large number of studies have shown that there are mutually coupled membrane components between the endoplasmic reticulum and mitochondria under fluorescence microscopy,called mitochondrial-endoplasmic reticulum membranes.The formation of high concentration of Ca2+ microdomains in this coupled region is closely related to calcium signaling regulation,mitochondrial morphology regulation,and endoplasmic reticulum stress.This coupling of physical structure makes ER stress and mitochondrial dysfunction affect each other.Therefore,researchers have speculated that mitochondrial dysfunction and endoplasmic reticulum stress together become one of the key factors in the pathogenesis of diabetic kidney disease.But at present,there is less research on this field.The recent discovery of the mitochondrial-endoplasmic reticulum structure coupling has provided new ideas for exploring the pathogenesis of diabetic kidney disease.At the same time,in previous studies,we have found that Chinese herbal medicine for supplementing qi and nourishing yin has certain positive influence on the endoplasmic reticulum stress of kidney tissue and renal parenchymal cells in the(4)HBZY-1 cells were divided into normal group,model group(TG group)and administration group(TG and Shengmai injection combined group),normal group was cultured with conventional culture medium,and model group was treated with 0.1 ?M TG.For stimulation,the treated group was stimulated with 0.1 ?M TG and 6 ?l/ml Shengmai Injection.After cultured for 24 hours in each group,the mitochondria and endoplasmic reticulum of the normal group,the model group,and the administration group were observed by confocal laser scanning microscopy.Structural changes were made to determine whether Shengmai Injection plays a protective role in endoplasmic reticulum and mitochondria in HBZY-1 cells under endoplasmic reticulum stress.(5)The expression of GRP78 gene and protein in HBZY-1 cells was detected by Real-time PCR and Western Blot after 24 hours in order to clarify the effect of Shengmai injection on endoplasmic reticulum.Whether HBZY-1 cells under stress have endoplasmic reticulum stress has a regulatory effect.(6)The results of Real-time PCR and Western Blot showed that compared with the normal group,the effect of 0.1 ? M TG on HBZY-1 cells for 24h induced the up-regulation of Mfnl gene and protein expression in mitochondrial fusion,and the difference was statistically significant(P<0.05).<0.05);and 6 ?l/ml Shengmai injection can significantly reduce the Mfnl gene and protein expression levels,the difference was statistically significant(P<0.05).However,for Mfn2,the expression of Mfn2 mRNA can only be changed by the stimulation of the model drug and intervention of therapeutic drugs(ie,the expression of mRNA of Mfn2 is up-regulated after the model is established,and the expression is decreased after intervention of Shengmai injection,the difference is statistically significant.Meaning);but no effect on the protein expression of Mfn2.Result:(1)The results of CCK-8 showed that the concentration of 0.2?M and 0.4?M early stages of diabetic kidney disease.Among them,Shengmai Yin is one of the typical prescriptions for Qi and Yin,and it is often used clinically to treat diabetes and diabetic kidney disease.Objective:To investigate the effect of Shengmai Yin on the mitochondria-endoplasmic reticulum structure in rat glomerular cells(HBZY-1)under endoplasmic reticulum stress,using the classic prescription of replenishing qi and nourishing yin,Shengmaiyin,as an intervention drug.The effects of associated proteins are expected to clarify their specific mechanisms of action and provide a reference for better guidance of the clinic and to find more drugs for renal protection.METHODS:The morphological changes of mitochondria and endoplasmic reticulum in rat glomerular mesangial cells were investigated under the condition of endoplasmic reticulum stress using endoplasmic reticulum stress agonist tropothane(TG).And the effect on the gene and protein expression of glucose regulatory protein 78(GRP78),mitochondrial fusion protein 1(Mfnl)and mitochondrial fusion protein 2(Mfn2).The specific method is as follows:(1)Determine whether HBZY-1 cells have toxic effects after a certain period of intervention with different concentrations of stimulants(including TG and Shengmai injection)by CCK-8 method.(2)HBZY-1 cells were treated with 0.05?M,0.1?M,and 0.2?M TG for 24h,and the morphological changes of mitochondria and endoplasmic reticulum were observed by phase-contrast microscopy to clarify the morphology of endoplasmic reticulum and mitochondria in HBZY-1 cells.(3)Real-time PCR was used to detect the expression level of GRP78,and the effects of TG at different concentrations and at different times on the ER stress in HBZY-1 cells were studied to explore the correlation of ER stress in HBZY-1 cells.concentration of TG used in the experiment at 24h and 48h,and the absorbance of cells in the normal group were not statistically different(P>0.05);The difference between the concentration of 0.8?M and the concentration of 0.8?M in the normal group at 36h after the action of 36h was statistically significant(P<0.05).The concentration of 8?l/ml of Shengmai injection at different concentrations for 24h and 36h for the experiment was not different from that of the normal group(P>0.05),but it was at the same concentration for 36h(except for 8?l/ml)and 48h after the Shengmai injection.All differences(P<0.05).(2)The results of phase-contrast microscopy showed that the proliferation of HBZY-1 cells was inhibited to a certain extent with increasing concentrations of HBZY-1 cells treated with 0.05?M,0.1 ?M and 0.2 ?M TG for 24 h,and the intracellular mitochondria and endoplasmic reticulum structure were changed.The boundary is blurred,especially the structure of the endoplasmic reticulum changes significantly.(3)Real-time PCR results showed that 0.05?M,0.1 pM,and 0.2?M TG could induce ER stress indicators GRP78 in HBZY-1 cells 24h,36h and 48h(except 0.05?M concentration,48h).The mRNA expression was up-regulated,and there was a certain concentration and time dependence.The difference was statistically significant(P<0.05).However,if the concentration of TG is too high,it can directly cause cell death,which in turn reduces the mRNA expression of GRP78.(4)Laser confocal microscopy results showed that compared with the normal group,0.1 ?M TG acting on HBZY-1 cells for 24h could cause the destruction of the endoplasmic reticulum and mitochondrion structure in the cells;while giving 6?l/ml Shengmai injection can Significantly improve the destruction of endoplasmic reticulum and mitochondrial structure,to a certain degree to protect the endoplasmic reticulum and mitochondrial structure.(5)Real-time PCR and Western Blot results showed that compared with the normal group,0.1?M TG on HBZY-1 cells for 24h could induce the expression of the endoplasmic reticulum stress index GRP78 gene and protein,the difference was statistically significant(P<0.05);The administration of Shengmai injection of 6 ?l/ml significantly decreased the expression of GRP78 gene and protein,and the difference was statistically significant(P<0.05).(6)The results of Real-time PCR and Western Blot showed that compared with the normal group,the effect of 0.1?M TG on HBZY-1 cells for 24h could induce the expression of genes and proteins of mitochondrial fusion-related indicators Mfnl and Mfn2,and the difference was statistically significant.(P<0.05);The injection of 6 ?l/ml Shengmai Injection significantly decreased the expression of Mfnl and Mfn2 gene and protein,and the difference was statistically significant(P<0.05).Conclusions:(1)Shengmai injection has a certain inhibitory effect on the proliferation of HBZY-1 cells,and has a certain time-dependent and dose-dependent effect.(2)Shengmai injection can improve the destruction of endoplasmic reticulum and mitochondria in HBZY-1 cells under endoplasmic reticulum stress,and it plays a role in protecting endoplasmic reticulum and mitochondrial structure to some extent.(3)At a certain concentration and within a certain period of time,TG can induce endoplasmic reticulum stress in HBZY-1 cells in a concentration-dependent manner.However,if the concentration of TG is too high,it can directly cause cell death,but can not induce endoplasmic reticulum stress.(4)Shengmai injection can significantly improve the endoplasmic reticulum stress level of HBZY-1 cells under endoplasmic reticulum stress conditions,and regulate the mitochondrial fusion level,and is concentration-dependent in a certain concentration range. |
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